Two clinical trials were performed in humans to treat hemophilia B using a recombinant adeno associated virus vector (rAAV) to express human Factor IX (hF.IX) from either skeletal muscle or liver. Patients with liver directed gene transfer transiently expressed therapeutic levels of hF.IX . Loss of hF.IX expression correlated with a transient elevation in transaminases, leading to elimination of hF.IX expressing hepatocytes. Follow up studies concluded that a cytotoxic T lymphocyte (CTL) response against an AAV2 capsid protein lead to the elimination of transduced hepatocytes expressing hF.IX. Since rAAV vectors do not express viral proteins, the levels of capsid protein are dependent on the input viral dose. Current treatment regimens for hemophilia B rely on administration of clotting factor concentrates derived either from donor plasma or recombinant protein. High costs and risks associated with infection with blood borne pathogens preclude prophylactic treatment and put patients at risk from developing complications from bleeding episodes. Gene transfer of hF.IX using a recombinant AAV vector has produced long term therapeutic levels of hF.IX in animal models of hemophilia. Data from the human clinical trial demonstrate a therapeutic benefit from AAV gene transfer of hF.IX. We have observed that tyrosine to phenylalanine mutations introduced into the AAV2 capsid protein block ubiquination and proteasomal degradation, resulting in improved gene transfer efficiency in mice and in human cells. We will investigate if these capsid mutants provide therapeutic hF.IX expression at lower viral doses and have a reduced risk of developing an immune response against the capsid protein. Since liver gene delivery via the portal vein is invasive, we will compare efficacy of intravenous viral delivery via the peripheral circulation versus portal vein injection. Murine and canine hemophilia B disease models will be used to determine the minimal viral dose required for therapeutic expression of hF.IX or canine Factor IX (cF.IX) using an AAV vector with the best performing AAV2 capsid mutations. PUBLIC HEALTH RELEVANCE: Immunological studies will be conducted to compare tolerance induction and maintenance, and immune response against both F.IX and AAV capsid proteins between wtAAV2 and the best performing AAV2 tyrosine capsid mutant. The improved gene transfer efficiency obtained from the novel tyrosine capsid mutations may prevent an immune response against the AAV2 capsid protein by reducing the viral dose (capsid protein) required for therapeutic efficacy.